DESCRIPTION: Carbonic anhydrase is a zinc-containing enzyme which catalyzes the reversible hydration of CO and is ubiquitous in humans where the enzyme participates in an assortment of metabolic functions. It facilitates in the liberation of CO stored in the form of HCO3- as in red cells and in the diffusion of CO2 across membranes. It is also involved in the formation of secretory fluids such as ocular fluid; indeed, carbonic anhydrase inhibitors are used to treat glaucoma. Past reports of carbonic anhydrase in procaryotes have been rare and largely confined to phototrophs where enzyme supplies CO2 for photosynthesis. Recently, a new class of carbonic anhydrase (the g class) was discovered in the strictly anaerobic methane-producing archaeon Methanosarcina thermophila with a crystal structure strikingly distinct from the well-characterized human enzymes. It is hypothesized that the g carbonic anhydrases have novel roles in the physiology of diverse nonphotosynthetic aerobes and anaerobes from the Archaea and Bacteria domains. The broad, long-term objective of this project is to determine the distribution and physiological function of carbonic anhydrases in diverse procaryotes. This application has three specific aims: (1) Ascertain the occurrence of g carbonic anhydrases in selected physiologically and phylogenetically diverse procaryotes. (2) Determine the physiological function of the carbonic anhydrase from M. thermophila in the pathway of acetate fermentation to methane and CO2. (3) Determine the catalytic mechanism of the prototypic g carbonic anhydrase from M. thermophila. These goals will be achieved by integrating genetic, biochemical and biophysical research methods. The results are expected to: (i) better assess the extent of carbonic anhydrases in procaryotes, (ii) identify novel physiological functions for carbonic anhydrases in nature, and (iii) provide new insights into the catalytic mechanism of all carbonic anhydrases and the g class in particular.